Chimeric poly peptides and the therapeutic use thereof against a flaviviridae infection

ABSTRACT

The invention relates to building a chimeric polypeptide used for preventing or treating a Flaviviridae infection. The use of the inventive chimeric polypeptide for producing recombinant viral vectors such as a measles living viral vector is also disclosed.

FIELD OF THE INVENTION

The present invention relates to the construction of a chimericpolypeptide for use in the prevention or treatment of a Flaviviridaeinfection. The present invention also relates to the use of the chimericpolypeptide in the production of recombinant viral vectors, such as alive measles viral vector to act as a vaccine.

BRIEF DESCRIPTION OF THE PRIOR ART

Flaviviruses (the Flaviviridae family) are envelope viruses the genomeof which is a RNA molecule with a positive polarity. The flavivirion iscomposed of three structural proteins designated C (core protein), M(membrane protein) and E (envelope protein). The E protein, which isexposed on the virus surface, is responsible for the principalbiological functions of the virion, including attachment of the virusand specific membrane fusion. Translation of genomic RNA produces aprecursor polyprotein which is simultaneously cleaved by cellular andviral proteases to generate individual viral proteins: three structuralproteins C, prM (the glycosylated precursor of the M protein), E, andseven non-structural proteins NS1 to NS5. Replication of flavivirusesoccurs in the cytoplasm of infected cells. Flaviviruses have complexnatural transmission cycles which involve several natural hosts (mainlymammals and birds) and vectors, the latter being hematophage arthropodssuch as ticks and mosquitoes. These viruses are the principal cause ofsevere human diseases such as hemorrhagic manifestations ormeningo-encephalitic syndromes. The dengue virus (DEN) is one of the twomajor flaviviruses known to be the cause of severe hemorrhagic diseasesacross the world.

Dengue is an emerging disease and a bane to public health. Developmentof live-attenuated vaccine candidates against the four serotypes of thedengue virus has been carried out for more than 50 years. The search fora vaccine is hindered by the absence of an animal model which mimics thedisease caused by the dengue virus, namely the hemorrhagic fever ofdengue and the shock syndrome associated therewith. Vaccineeffectiveness can be evaluated in the mouse using a neurovirulent strainof DEN virus adapted to the mouse, which kills adult animals afterintracerebral inoculation. However, studies based on that type of murinemodel do not reflect the activity of the dengue virus in man. In themonkey, protection can only be demonstrated by measuring the reductionin viremia after an experimental infectious challenge. Thus, theultimate test for a vaccine against dengue must be based on clinicaltrials carried out in man. The majority of attenuated vaccine candidateswhich have been developed over the last 50 years have been either tooreactogenic or insufficiently immunogenic in clinical trials. Severalattenuated tetravalent candidates are currently in phase I or IIclinical trials. The difficulty in the development of this type oflive-attenuated tetravalent vaccines appears to be in obtaining abalanced mixture of four valencies in order to generate protectiveimmunity against the four serotypes.

Various live chimeric vaccine candidates are also being developed. Theyare based on the exchange of homologous structural genes betweendifferent flaviviruses. Several types of intertype chimeric viruses havebeen constructed and tested in the mouse or monkey. Chimeric viruseshave been constructed with Chimerivax® techniques based on the yellowfever vaccinal virus (YF-17D). Tetravalent mixtures of those YF-17D/DENchimeras have been tested in the rhesus monkey. Here again, the balancebetween the four chimeric viruses is difficult to obtain for uniformimmunization. A phase I/II clinical test is currently being prepared totest a chimerivax-dengue 2 vaccine. Other approaches include subunitcandidate vaccines produced from an expression vector, and naked DNAtype candidates which are in pre-clinical development. Thus, thedevelopment of a tetravalent vaccine against the dengue virus is stillan unresolved problem, one of the most important on the list of theWorld Health Organization.

Thus, there is a need to develop novel effective vaccine candidateswhich are easy to produce and to formulate, for the prevention ortreatment of an infection by a virus of the Flaviviridae family such asthe dengue virus.

SUMMARY OF THE INVENTION

The present invention relates to a chimeric polypeptide and itsapplications in the prevention or treatment of an infection byFlaviviridae.

More particularly, an object of the present invention is a chimericpolypeptide comprising a peptide of a subdomain the E protein ofFlaviviridae bound to a peptide of a subdomain of the Membrane M proteinof Flaviviridae.

An object of the present invention is also an isolated or purifiedpolynucleotide coding for a chimeric polypeptide of the invention.

An object of the present invention is also a recombinant measles viralvector into the genome of which a polynucleotide of the invention hasbeen inserted.

An object of the invention also pertains to purified monoclonal orpolyclonal antibodies specifically recognizing at least onepolynucleotide as defined above and/or the chimeric polypeptide of theinvention.

The invention also relates to the use of a viral vector of the inventionfor the preparation of an immunogenic composition intended for theprevention or treatment of a Flaviviridae infection in a sensitivespecies.

The present invention also relates to cloning or expression vectorscomprising a polynucleotide of the invention.

Another object of the present invention is to provide an immunogeniccomposition intended for the prevention and/or treatment of aFlaviviridae infection in a sensitive species, characterized in that itcomprises at least one of the following elements:

-   -   a chimeric polypeptide as defined above;    -   a polynucleotide according to the invention;    -   a recombinant viral vector according to the invention;    -   an antibody according to the invention; and    -   a cloning and/or expression vector according to the invention.

The present invention also proposes a method for preventing and/ortreating a Flaviviridae infection in a sensitive species, comprisingadministering a pharmaceutically effective amount of at least one of thefollowing elements:

-   -   a chimeric polypeptide as defined above;    -   a polynucleotide according to the invention;    -   a recombinant viral vector according to the invention;    -   an antibody according to the invention; and    -   a cloning and/or expression vector according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the 384 nucleotide (nt) sequence coding for the[EDIII]_(DV-1) domain of the

FGA/89 viral strain fused via the Arg-Ser dipeptide to the signalpeptide of calreticulin (ssCRT) flanked by the two BsiWI (5′) and BssHII(3′) sites necessary for insertion into the MV_(Schw) vector;

FIG. 2 shows the 516 nucleotide (nt) sequence coding for the[EDIII+M¹⁻⁴⁰]_(DV-1) fusion protein of the FGA/89 viral strain fused viathe Arg-Ser dipeptide to the signal peptide of calreticulin (ssCRT)flanked by the two BsiWI (5′) and BssHII (3′) sites necessary forinsertion into the MV_(Schw) vector;

FIG. 3 shows Vero cells infected with recombinant measles viral vectorsaccording to the invention. Multiplicity of infection 10 TCIP50/cell for30 h. Immunofluorescence carried out with a specific anti-DV-1 HMAFantibody;

FIG. 4 shows the expression and secretion of the [EDIII]_(DV-1) and[EDIII+M¹⁻⁴⁰]_(DV-1) antigenic domains in cytoplasmatic lysates (C) andsupernatants (S) from Vero cells infected with recombined MVSchw-DV-1viruses;

FIG. 5 shows the expression and secretion of the [EDIII]_(DV-1) and[EDIII+M¹⁻⁴⁰]_(DV-1) antigenic domains in filtered and concentratedsupernatants from drosophila S2 cells inducibly expressing theseantigens;

FIG. 6 shows a nucleotide sequence coding for a chimeric polypeptideaccording to a preferred mode of the invention;

FIG. 7 shows an amino acid sequence of a chimeric polypeptide accordingto a preferred mode of the invention and the nucleotide sequence codingfor it;

FIG. 8 shows an amino acid sequence of a dimer of the ectodomain III(EDIII) according to a preferred mode of the invention, and thenucleotide sequence coding for it;

FIG. 9 shows an amino acid sequence of a dimer of the ectodomain III(EDIII) according to a preferred mode of the invention, and thenucleotide sequence coding for it;

FIG. 10 shows an amino acid sequence of a chimeric polypeptide accordingto a preferred mode of the invention, and the nucleotide sequence codingfor it;

FIG. 11 shows an amino acid sequence of the ectodomain III (EDIII)according to a preferred mode of the invention, and the nucleotidesequence coding for it;

FIG. 12 shows an amino acid sequence of a dimer of the ectodomain III(EDIII) according to a preferred mode of the invention, and thenucleotide sequence coding for it;

FIG. 13 shows a nucleic acid sequence coding for a peptide of theectodomain III (EDIII) peptide according to a preferred mode of theinvention;

FIG. 14 shows a nucleotide sequence coding for a chimeric polypeptideaccording to a preferred mode of the invention;

FIGS. 15 to 19 show an amino acid sequence of chimeric polypeptidesaccording to a preferred mode of the invention;

FIGS. 20 to 24 show a nucleotide sequence coding respectively for thechimeric polypeptides of FIGS. 15 to 19;

FIGS. 25 to 29 respectively show a representative diagram of thechimeric polypeptides of FIGS. 15 to 19;

FIG. 30 shows the peptide sequence of the apoptoM sequence of fourserotypes of the dengue virus used in the construction of a chimericprecursor according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The originality of the present invention is based on the construction ofa chimeric polypeptide and its applications in the prevention ortreatment of an Flaviviridae infection. The term “Flaviviridae” means avirus selected from the group constituted by the West Nile virus, denguevirus, Japanese encephalitis virus and yellow fever virus.

1. Polypeptide and Polynucleotide

In a first aspect, the present invention relates to a chimericpolypeptide comprising a peptide of a subdomain of the E protein ofFlaviviridae linked to a peptide of a subdomain of the membrane Mprotein of Flaviviridae. The invention also pertains to a polypeptidewhich consists of said peptides.

The term “polypeptide” as used in the present invention means bothproteins and peptides. By “chimeric polypeptide” it is meant any proteinor peptide comprising sub-portions of different origins, for example apolypeptide A deriving from a first species and a polypeptide (proteinor peptide) deriving from a second species. A protein or peptide is alsoconsidered to be a chimeric polypeptide if it includes sub-portionsderiving from different proteins or peptides from the same species, oreven from the same protein or peptide from different species.

Preferably, the peptide of a subdomain of the E protein consists of theectodomain III comprising an amino acid sequence as defined in any oneof the following sequences:

-   -   amino acids 19 to 120 of SEQ ID NO: 1;    -   amino acids 19 to 119 of SEQ ID NO: 3;    -   amino acids 21 to 123 of SEQ ID NO: 6;    -   amino acids 21 to 121 of SEQ ID NO: 12; and    -   amino acids 21 to 123 of SEQ ID NO: 14.

More particularly, the peptide of a subdomain of the E protein consistsof

a dimer of the ectodomain III of the dengue 1, 2, 3 or 4 viruscomprising an amino acid sequence as defined in any one of the followingsequences:

-   -   amino acids 21 to 262 of SEQ ID NO: 8; and    -   amino acids 21 to 262 of SEQ ID NO: 10;

or a tetramer of the ectodomain III of the dengue 1, 2, 3, 4 viruscomprising a sequence ranging from amino acid 21 to 494 of SEQ ID NO: 16or ranging from amino acid 18 to 429 of SEQ ID NO: 24.

Regarding the peptide of a subdomain of the M protein, this inparticular consists of the ectodomain 1-40 comprising an amino acidsequence ranging from position 123 to 170 of SEQ ID NO: 3 or in theapoptoM sequence comprising an amino acid sequence ranging from position154 to 170 of SEQ ID NO: 3 or ranging from position 122 to 132 of SEQ IDNO: 12.

In a preferred mode, the chimeric polypeptide of the invention furthercomprises a binding segment binding the peptide of a subdomain of the Eprotein to the peptide of a subdomain of the M protein. Said bindingsegment is preferably a pentapeptide with sequence: RRDKR or RREKR.

Highly preferably, the chimeric polypeptide of the invention comprisesan amino acid sequence as defined in any one of the following sequences:

a) amino acids 19 to 172 of SEQ ID NO: 3;

b) amino acids 21 to 168 of SEQ ID NO: 6;

c) amino acids 21 to 132 of SEQ ID NO: 12;

d) amino acids 18 to 624 of SEQ ID NO: 20

e) amino acids 18 to 609 of SEQ ID NO: 21;

f) amino acids 18 to 624 of SEQ ID NO: 22;

g) amino acids 18 to 489 of SEQ ID NO: 23; and

h) amino acids 21 to 474 of SEQ ID NO: 24.

The invention also relates to polypeptides (and fragments thereof) whichare coded by the nucleotide sequences mentioned below.

Highly preferably, the polypeptide of the invention has a percentageidentity of at least 80% after optimum alignment with a sequence asdefined in any one of amino acid sequences a) to h) defined above,preferably at least 90%, more preferably at least 98% and still morepreferably at least 100%.

In a connected aspect, the invention relates to an isolated or purifiedpolynucleotide coding for a chimeric polypeptide as defined above.

By “isolated or purified”, it is meant molecules which have been alteredby man from their native state, i.e. if such molecules exist in nature,they have been changed and/or removed from their initial environment. Asan example, a polynucleotide or a polypeptide naturally present andfound in the biological environment of a living organism which naturallyexpresses it is not “isolated” in this context. However, the samepolynucleotide or polypeptide when separated from its naturalenvironment and/or obtained by cloning, amplification and/or chemicalsynthesis is considered in the present invention to be “isolated”.Further, a polynucleotide or polypeptide which is introduced into anorganism by transformation, gene manipulation or any other recombinationmethod is “isolated” even if it is present in said organism.

By the terms “nucleotide sequence”, “nucleic acid”, “nucleic sequence ornucleic acid sequence”, “polynucleotide”, oligo nucleotide”,“polynucleotide sequence”, which will be used indiscriminately in thepresent description, it is meant a precise chain of nucleotides, whichmay or may not be modified, which allows to define a fragment or aregion of a nucleic acid, which may or may not comprise non-naturalnucleotides and which can correspond both to a double-stranded DNA, asingle-stranded DNA or to transcription products of said DNAs. Thus, thenucleic sequences of the invention also encompass PNAs (peptide nucleicacid) or the like. The polynucleotide fragments of the inventioncomprise at least 15 consecutive nucleotides. Preferably, they compriseat least 20 consecutive nucleotides and more preferably they comprise atleast 30 consecutive nucleotides.

The invention also pertains to fragments of polynucleotides, whichconsist of fragments of 15, 20 or 30 successive nucleotides.

Any polynucleotide which has been chemically, enzymatically ormetabolically modified but which has retained the biochemical propertiesof the original chimeric polypeptide is included within the scope of thepresent invention.

In a preferred embodiment, the polynucleotide of the present invention,when it codes for a chimeric polypeptide of the invention,advantageously comprises a nucleotide sequence as defined in any one ofthe following sequences:

a) nucleotides 7 to 492 of SEQ ID NO: 4;

b) SEQ ID NO: 5;

c) Nucleotides 7 to 504 of SEQ ID NO: 7;

d) Nucleotides 7 to 504 of SEQ ID NO: 13; and

e) SEQ ID NOS: 25 to 29.

Highly preferably, the polynucleotide of the invention has a percentageidentity of at least 80% after optimum alignment with a sequence asdefined in any one of the nucleotide sequences a) to e) defined above,preferably at least 90%, more preferably at least 98% and mostpreferably at least 100% identity.

The term “percentage identity” between two nucleic acid or amino acidsequences as used in the present invention means a percentage ofidentical nucleotides or amino acid residues between the two sequencesto be compared, obtained after best alignment, that percentage beingpurely statistical and the differences between the two sequences beingrandomly distributed and over their entire length. By “best alignment”or “optimum alignment”, it is meant the alignment at which thepercentage identity determined as below is the highest. Sequencecomparisons between two nucleic acid or amino acid sequences aretraditionally carried out by comparing these sequences after havingaligned them in an optimum manner, said comparison being carried outusing comparison segments or windows to identify and compare localregions with sequence similarity. The optimum sequence alignment forcomparison may be carried out manually or using a Smith and Waterman(1981) local homology algorithm, using the Neddleman and Wunsch (1970)local homology algorithm, using the Pearson and Lipman (1988) sequencesimilarity search method, or using software employing these algorithms(GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr,Madison, Wis.). To obtain an optimal alignment, the BLAST program ispreferably used, with the BLOSUM 62 matrix. It is also possible to usePAM or PAM250 matrices.

The percentage identity between two nucleic acid or amino acid sequencesis determined by comparing these two sequences aligned in an optimummanner, the nucleic acid or amino acid sequence to be compared possiblycomprising additions or deletions compared with the reference sequencefor optimum alignment between these two sequences. The percentageidentity is calculated by determining the number of identical positionsfor which the nucleotide or amino acid residue is identical between thetwo sequences, dividing this number of identical positions by the totalnumber of compared positions and multiplying the result obtained by 100to obtain the percentage identity between these two sequences.

The term “nucleic sequences having a percentage identity of at least80%, preferably at least 90%, more preferably at least 98% after optimumalignment with a reference sequence”, is intended to designate nucleicsequences having, with respect to the reference nucleic acid, certainmodifications, in particular a deletion, truncation, extension, chimericfusion, and/or a substitution, in particular a point substitution, andfor which the nucleic sequence has at least 80%, preferably at least90%, more preferably at least 98% identity after optimum alignment withthe reference nucleic sequence. Preferably, the specific hybridizationconditions or high stringency conditions will be such that they ensureat least 80%, preferably at least 90%, more preferably at least 98%identity after optimum alignment between one of the two sequences andthe complementary sequence of the other.

A hybridization under high stringency conditions means that thetemperature and ionic strength conditions are selected so that they canmaintain the hybridization between two complementary nucleic acidfragments. By way of illustration, the highly stringent conditions forthe hybridization step with the aim of defining the nucleotide sequencesdescribed above are advantageously as follows.

DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)pre-hybridization at 42° C. for 3 hours in a phosphate buffer (20 mM, ph7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 MNaCl+0.015 M of sodium citrate), 50% formamide, 7% sodium dodecylsulphate (SDS), 10× Denhardt's solution, 5% dextran sulphate and 1%salmon sperm DNA; (2) hybridization proper for 20 hours at a temperaturedepending on the probe size (i.e.: 42° C. for a probe size >100nucleotides), followed by 2 washings of 20 minutes at 20° C. in 2×SSC+2%SDS, 1 washing of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS. The finalwashing is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. fora probe size >100 nucleotides. The high stringency hybridizationconditions described above for a polynucleotide of a defined size may beadapted by the skilled person for larger or smaller oligonucleotides, asdiscussed by Sambrook et al, 1989.

The polypeptides and polynucleotides of the present invention may beprepared using any appropriate process. They may in particular beobtained by chemical synthesis, but it is also possible to obtain themvia a biological route pathway, especially using different vectors inappropriate cell cultures. The peptides of the present invention may, ifnecessary, be in the deglycosylated or glycosylated form. A personskilled in the field of the invention will be able to obtain differentpolynucleotides/polypeptides and will also be able to determine which ofthe polynucleotides/polypeptides obtained have a suitable biologicalactivity.

2. Recombinant Viral Vector

In a further aspect, the invention concerns a recombinant measles viralvector with a polynucleotide of the invention inserted into the genomethereof. Such vectors are prepared using methods which are routinelyused by the skilled person and the resulting clones may be introducedinto a suitable host using standard methods which are known to theskilled person.

In a preferred mode, the recombinant viral vector of the invention isadvantageously a live measles (Schwarz strain) viral vector, such asthose selected from the group of viral vectors constituted by thosedeposited at the CNCM under numbers I-3440, I-3442, I-3452, I-3453,I-3454, I-3455, I-3619, I-3620, I-3621, I-3622 and I-3623. The completeantigenome sequence of the measles Schwarz strain virus can, forexample, be obtained from pTM plasmids of strains deposited undernumbers I-3440 and I-3442.

The vector pTM-MVSchw2-[EDIII+M¹⁻⁴⁰]WNV(IS-98-ST1) was deposited at theCNCM (Paris, France) on 26 May 2005 under number 1-3440.

This vector is a pTM plasmid containing the complete antigenome of themeasles virus, Schwarz strain, and an additional expression unit placedbetween the P and M genes containing the sequence of the domain III ofthe envelope E protein of West Nile virus (WNV IS-98-ST1) fused to thesequence 1-40 of the membrane protein.

The vector may be cultivated in 100 μ/ml LB ampicillin medium afterinoculating small colonies.

It is preserved long-term by freezing at −80° C.

The vector pTM-MVSchw2-[EDIII+ApoptoM]DV-1(FGA89) was deposited at theCNCM (Paris, France) on 26 May 2005 under number I-3442.

This vector is a pTM plasmid containing the complete antigenome of themeasles virus, Schwarz strain, and an additional expression unit placedbetween the P and M genes containing the sequence of the domain III ofthe envelope E protein of the dengue 1 virus, FGA89 strain, fused to theapoptotic sequence of the Membrane M protein.

The vector may be cultivated in 100 μ/ml LB ampicillin medium afterinoculating small colonies.

It is preserved long-term by freezing at −80° C.

The vector pTRE₂-SSCRT-ApoptoMden1/DIII-Hα1-2 was deposited at the CNCM(Paris, France) on 16 Jun. 2006 under number 1-3452.

This vector is a pTRE₂ plasmid containing the sequence of the signalpeptide of human calreticulin fused to the pro-apoptotic sequences ofthe M protein and to the ectodomains III of the E protein with theα-helice sequences of the dengue 1 and 2 viruses.

The sequence of the insert contained in the plasmid and constituting apolynucleotide of the invention is SEQ ID NO: 9 shown in FIG. 8.

The vector may be cultivated in 100 μ/ml LB ampicillin medium.

It is preserved long-term by freezing at −80° C.

The vector pTRE₂-ssCRT-ApoptoMden1/DIII-Hα3-4 was deposited at the CNCM(Paris, France) on 16 Jun. 2006 under number I-3453.

This vector is a pTRE₂ plasmid containing the sequence of the signalpeptide of human calreticulin fused to the pro-apoptotic sequences ofthe M protein of dengue 1 virus and to the sequences of the ectodomainsIII and Ha of the dengue 3-4 viruses.

The sequence of the insert contained in the plasmid and constituting apolynucleotide of the invention is SEQ ID NO: 11 shown in FIG. 9.

The vector may be cultivated in LB ampicillin 100 μ/ml medium.

It is preserved long-term by freezing at −80° C.

The vector pTRE₂-ssCRT-ApoptoMden1/DIII-Hα1-2-3-4 was deposited at theCNCM (Paris, France) on 16 Jun. 2005 under number I-3454.

This vector is a pTRE₂ plasmid containing the signal peptide sequence ofhuman calreticulin fused to the pro-apoptotic sequences of the M proteinof dengue 1 virus and to the sequences of the ectodomains III with αhelices (Hα) of the E proteins of the dengue 1, 2, 3, 4 viruses.

The sequence of the insert contained in the plasmid and constituting apolynucleotide of the invention is SEQ ID NO: 4 shown in FIG. 2.

The vector may be cultivated in 100 μ/ml LB ampicillin medium.

It is preserved long-term by freezing at −80° C.

The vector pTRE₂-ssCRT-EctoM40den1/DIII-Hα1-2-3-4 was deposited at theCNCM (Paris, France) on 16 Jun. 2005 under number 1-3455.

This vector is a pTRE₂ plasmid containing the signal peptide sequence ofhuman calreticulin fused to the sequences of the ectodomain 1-40 of theM protein of the dengue 1 virus and to the sequences of the ectodomainsIII with α helices (Hα) of the E proteins of the dengue 1, 2, 3, 4viruses.

The sequence of the insert contained in the plasmid and constituting apolynucleotide of the invention is SEQ ID NO: 17 shown in FIG. 12.

The vector may be cultivated in 100 μ/ml LB ampicillin medium.

It is preserved long-term by freezing at −80° C.

The vector pUC57-TetraDVA (introduced into an E. coli strain) wasdeposited at the CNCM (Paris, France) on 14 Jun. 2006 under numberI-3619.

This vector is a pUC plasmid containing a nucleotide sequence optimizedfor the expression in mammalian cells of a tetrameric construct of theectodomains III of the envelope E protein of the 4 serotypes (1, 2, 3,4) of the dengue virus, fused to the ectodomain of the Membrane Mprotein.

The insert contained in the plasmid vector is shown in FIG. 25.

The strain containing the vector may be cultivated in 100 μ/ml LBampicillin medium.

The vector pUC57-TetraDVB (introduced into an E. coli strain) wasdeposited at the CNCM (Paris, France) on 14 Jun. 2006 under numberI-3620.

This vector is a pUC plasmid containing a nucleotide sequence optimizedfor the expression in mammalian cells of a tetrameric construct of theectodomains III of the envelope E protein of the 4 serotypes (1, 2, 3,4) of the dengue virus, fused to the ectodomain of the Membrane Mprotein.

The insert contained in the plasmid vector is shown in FIG. 26.

The strain containing the vector may be cultivated in 100 μ/ml LBampicillin medium.

The vector pUC57-TetraDVC (introduced into an E. coli strain) wasdeposited at the CNCM (Paris, France) on 14 Jun. 2006 under numberI-3621.

This vector is a pUC plasmid containing a nucleotide sequence optimizedfor the expression in mammalian cells of a tetrameric construct of theectodomains III of the Envelope E protein of the 4 serotypes (1, 2, 3,4) of the dengue virus, fused to the ectodomain of the Membrane Mprotein.

The insert contained in the plasmid vector is shown in FIG. 27.

The strain containing the vector may be cultivated in 100 μ/ml LBampicillin medium.

The vector pUC57-TetraDVD (introduced into an E. coli strain) wasdeposited at the CNCM (Paris, France) on 14 Jun. 2006 under numberI-3622.

This vector is a pUC plasmid containing a nucleotide sequence optimizedfor the expression in mammalian cells of a tetrameric construct of theectodomains III of the Envelope E protein of the 4 serotypes (1, 2, 3,4) of the dengue virus, fused to the ectodomain of the Membrane Mprotein.

The insert contained in the plasmid vector is shown in FIG. 28.

The strain containing the vector may be cultivated in 100 μ/ml LBampicillin medium.

The vector pUC57-TetraDVE (introduced into an E. coli strain) wasdeposited at the CNCM (Paris, France) on 14 Jun. 2006 under numberI-3623.

This vector is a pUC plasmid containing a nucleotide sequence optimizedfor the expression in mammalian cells of a tetrameric construct of theectodomains III of the Envelope E protein of the 4 serotypes (1, 2, 3,4) of the dengue virus, fused to the ectodomain of the Membrane Mprotein.

The insert contained in the plasmid vector is shown in FIG. 29.

The strain containing the vector may be cultivated in 100 μ/ml LBampicillin medium.

3. Cloning and/or Expression Vectors and Antibodies

In a further aspect, the invention concerns any cloning and/orexpression vector and any cell host (prokaryotic or eukaryotic)transformed by such a vector, and comprising regulation elementsallowing the expression of the nucleotide sequence coding for a chimericpolypeptide of the invention. Such vectors are prepared using methodswhich are routinely used by the skilled person, and the resulting clonesmay be introduced into an appropriate host using standard methods suchas, for example, lipofection, electroporation, thermal shock,transformation after chemical permeabilization of the membrane, or cellfusion.

The invention also encompasses host cells, in particular eukaryotic andprokaryotic cells, transformed by the vectors of the invention as wellas transgenic animals, preferably mammals, with the exception of man,comprising one of said transformed cells of the invention. These animalsmay be used as models, to study the etiology of inflammatory and/orimmune diseases, and in particular inflammatory diseases of thedigestive tract, or in the study of cancers.

Of the cells which may be used in the present invention, the followingmay be cited: bacterial cells (Olins and Lee (1993), Curr OpBiotechnology 4: 520), but also yeast cells (Buckholz (1993), Curr OpBiotechnology 4, 538), as well as animal cells, in particular mammaliancell cultures (Edwards and Aruffo (1993), Curr Op Biotechnology 4, 558).

In the context of the present invention, the term “cloning and/orexpression vector” refers to a polynucleotide construct designed to betransfected into different cell types. For this reason, these vectorsencompass expression vectors designed for the expression of a nucleotidesequence in a host cell; cloning vectors designed for the isolation,propagation and replication of inserted nucleotides or shuttle vectorswhich comprise the attributes of more than one vector.

4. Polyclonal or Monoclonal Antibodies

The polypeptides and polynucleotides of the present invention may alsobe used to prepared polyclonal or monoclonal antibodies capable ofbinding (preferably in a specific manner) to at least one chimericpolypeptide/polynucleotide of the invention. The present invention thusalso relates to such purified antibodies which may be obtained by verywell known techniques such as, for example, the technique described byKolher and Milstein (continuous cultures of fused cells secretingantibody of predefined specificity, Nature (1975), 262: 495-497). In apreferred embodiment of the invention, the antibodies are of the“humanized” type. A person skilled in the art would be able to use hisgeneral knowledge to prepare these types of antibodies.

5. Methods and Use

In a further aspect, the invention concerns the prevention and/ortreatment of a Flaviviridae infection in a sensitive species. Moreparticularly, the invention relates to the use of a recombinant viralvector of the invention for the preparation of an immunogeniccomposition intended for the prevention or treatment of a Flaviviridaeinfection in a sensitive species. The term “sensitive species” means anyanimal which is susceptible to a Flaviviridae infection, for example ahuman being.

The invention also relates to a method for preventing and/or treating aFlaviviridae infection in a sensitive species, comprising administeringa pharmaceutically effective amount of at least one of the followingelements:

-   -   a chimeric polypeptide of the invention;    -   a polynucleotide of the invention;    -   a recombinant viral vector of the invention;    -   an antibody of the invention; and    -   a cloning and/or expression vector of the invention.

The term “Flaviviridae infection” means, for example, flaviviroses suchas dengue, yellow fever, Japanese encephalitis and West Nile fever. Themeans for preparing and administering the elements of the presentinvention will not be described in more detail as they are already knownto the skilled person.

6. Compositions

The present invention also concerns immunogenic compositions useful inthe prevention and/or treatment of a Flaviviridae infection. The term“immunogenic composition” means a composition which contains elementshaving the capacity to induce, in vivo or in vitro, a cellular and/orhumoral type immune response.

In a preferred embodiment, the composition of the present inventionfurther contains a pharmaceutically acceptable vehicle and an elementselected from the group constituted by:

-   -   a polynucleotide of the invention;    -   a chimeric polypeptide of the invention;    -   a recombinant viral vector of the invention;    -   an antibody of the invention; and    -   a cloning and/or expression vector of the invention.

The compositions of the present invention may be in any solid or liquidform which is normal for pharmaceutical administration, examples offorms of administration being a liquid, a gel, or any other supportwhich can allow controlled release, for example. Examples ofcompositions which may be used which may be cited are compositions whichcan be injected into human beings.

The compositions of the invention may also comprise components whichincrease or are capable of increasing the immunogenicity of the chimericpolypeptides of the invention, in particular other immunogenic peptides,specific or non-specific immunity adjuvants such as alun, QS21, Freund'sadjuvant, SBA₂ adjuvant, montanide, polysaccharides or equivalentcompounds.

A person versed in the art will be able to prepare pharmaceuticallyacceptable compositions and to determine, as a function of severalfactors, the preferred mode of administration and the amount which hasto be administered. Factors which may influence the choice include: thenature of the treatment, the exact nature of the ingredients, active ornon active, in the composition; the stage of the disease; the condition,age and weight of the patient, etc.

EXAMPLES

The following examples demonstrate other characteristics and advantagesof the present invention and serve to illustrate rather than limit thescope of the present invention. Modifications and variations may be madewithout departing from the spirit and scope of the invention. Althoughother methods or products equivalent to those discussed below may beused to test or implement the present invention, preferred equipment andmethods are described.

Example 1 The Recombined Measles Virus MV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) asa Prototype for a Candidate Vaccine Against Dengue (PTR156)

Described in FIGS. 1 and 2, two immunogenic constructs based on the onehand on the capacity of domain III of the envelope E glycoprotein of thedengue virus to induce neutralizing antibodies and on the other hand onthe involvement of the ectodomain of the membrane M protein (M¹⁻⁴⁰) inviral pathogenicity (apoptosis) were inserted into the genome of theattenuated measles virus (Schwarz strain) (MVSchw). The viral sequenceswere under the dependency of the signal peptide of calreticulin to allowthem to be targeted into the secretion pathway. The expression of the[EDIII]_(DV-1) and [EDIII+M¹⁻⁴⁰]_(DV-1) sequences by the recombinedviruses MVSchw was verified in infected Vero cells by indirectimmunofluorescence using a polyclonal HMAF mouse serum directed againstDV-1 (FIG. 3). Secretion of the EDIII and EDIII antigenic domains fusedto the M¹⁻⁴⁰ sequence (with the intercalating pentapeptide RRDKR) of thedengue virus, type-1 (DV-1) of the FGA/89 strain (Holmes E C et al, MolBiol Evol 16(3): 405-409, 1999 and Despres P et al, Virology 196:209-219) was observed by Western blot analysis in the supernatants ofcells infected with the MV_(Schw)-[EDIII]_(DV-1) andMV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) virus respectively (FIG. 4). Similarly,the production and secretion in the culture supernatants of aS2/[EDIII+M^(1-40]) _(DV-1) drosophila cell line induced for expressionof these proteins has been demonstrated (FIG. 5).

Two groups of four adult CD46/FNAR mice were immunized with 10⁴ TCID50of each of the recombined MVSchw viruses (Tables 1 and 2). The emptyMVSchw virus was used as a negative control. We determined theproduction of antibodies directed against the MVSchw virus (MV Ag) andthe DV-1 virus (DV-1 Ag) including those which were specific to EDIII(Thullier P et al, 2001, J Gen Virol 82: 1885-1892) and neutralizing(Anti-DV-1 FRNT75) in immunized mice (Tables 1 and 2). We observed thatthe MV_(Schw)-[EDIII]_(DV-1) virus is less effective at producingspecific antibodies to DV-1 or EDIII alone after two inoculations spacedby one month (Table 1). Using the same immunization protocol, weobserved that the MV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) virus inducedsignificant titers of antibodies directed against DV-1 and against EDIIIalone (Table 2). Anti-DV-1 antibodies including those which wereneutralizing were still detected four months after the end ofimmunization.

When mice immunized more than 6 months previously withMV_(Schw)-[EDIII+^(M1-)40]_(DV-1) were given a booster with a singledose of 5 μg of total proteins of a concentrate of supernatant ofS2/[EDIII+M¹⁻⁴⁰]_(DV-1) drosophila cells induced for expression of thefusion protein [EDIII+M¹⁻⁴⁰]_(DV-1) and in the presence of Alugel as anadjuvant, large amounts of anti-DV-1, anti-EDIII and neutralizing DV-1antibodies were observed, which means that there was a well establishedhumoral memory response in the vaccinated animals (Table 2). Theanti-DV-1 antibodies were still present several months after theantigenic booster (Table 2).

Three months after the antigenic booster with r[EDIII+M¹⁻⁴⁰]_(DV-1),mice immunized with MV_(schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) were inoculatedintraperitoneally with 10⁷ FFU of FGA/NA dld strain DV-1 (DV-1 isresponsible for an asymptomatic infection in the IFNAR mouse). Largetiters of anti-DV-1 antibody (in particular directed against EDIII)including those which neutralized DV-1 Hawaii strain (neutralizing titer# 4000) were observed after 3 weeks of viral challenge (Table 2). Itshould be noted that mice immunized 9 months previously withMV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) (this construct not inducing anti-EDIIIantibody or neutralizing DV-1 antibody) then challengedintraperitoneally with 10⁷ FFU of FGA/NA did strain DV-1 producedanti-DV-1, anti-EDIII and neutralizing titers of 20000, 5000 and 80respectively; these were values equivalent to those observed in BALB/cmice or IFNAR mice challenged under the same experimental conditions.

In conclusion, the fusion sequence [EDIII+M¹⁻⁴⁰]_(DV-1) secreted by theMV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) virus is capable of generatingneutralizing anti-DV-1 antibodies and of inducing a long term humoralmemory response which is effectively stimulated on the one hand by thesoluble antigen r[EDIII+M¹⁻⁴⁰]_(DV-1) and on the other hand in responseto a viral infection. In contrast, the only antigenic EDIII domainsecreted by the MV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) virus is of lowimmunogenicity in CD46⁺-FINAR mice. Our complementary work demonstratesthat the pro-apoptotic ApoptoM (M³²⁻⁴⁰)peptide at the C-terminal end ofM determines the immunogenic power of the [EDIII+^(M1-)40]_(DV-1) fusionsequence since the MV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) virus without ApoptoMinduces a production of anti-DV-1 antibody equivalent to that obtainedafter immunization with the MV_(Schw)-[EDIII]_(DV-1) virus.

As a general methodology for pediatric vaccination against dengue, wepropose to immunize young individuals with theMV_(Schw)-[EDIII+M¹⁻⁴⁰]_(DV-1) virus using a double inoculation spacedby one month, then to restimulate them later with the solubler[EDIII+M¹⁻⁴⁰]_(DV-1) antigen as a vaccine booster or prophylacticallyagainst the risk of an infection by the dengue virus. This immunizationstrategy is being validated for the four serotypes of dengue based on anantigen composed of a tetramer of the four EDIII domains of DV-1, -2, -3and -4 fused to the cytotoxic ApoptoM sequence.

Our first experimental results underline the importance of a reducedsize immunogen derived from the envelope protein of the DV-1 virus incombination with the immunostimulating capacity of the live measlesvector, a strategy which allows the induction of a neutralizing humoralresponse which is effective against the dengue virus. They define aproof of concept for the design of the tetrameric constructs of theantigenic domains of DV which will allow simultaneous and long termimmunization against the four dengue serotypes.

Example 2 Construction of Chimeric Polypeptides Optimized for Expressionin Mammals

In the present example, the inventors have developed novel antigenicconstructs useful against a Flaviviridae infection.

These novel chimeric polypeptides are based on an antigen composed of atetramer of the four EDIII domains of the four serotypes of the denguevirus, fused together and to one or the other of the cytotoxic apoptoMsequences shown in FIG. 30.

Thereafter, these chimeric polypeptides optimized for expression in amammal were inserted into the genome of the attenuated measles virus,Schwarz strain, (MVSchw) viral strain. FIGS. 15 to 29 show the aminoacid sequences of five preferred chimeric polypeptides and thepolynucleotides encoding them.

These chimeric polypeptides were constructed using the followingoptimization conditions:

Optimization information for these sequences:

-   -   (Cai No=0.806, mean % GC=53.46, GC distribution: homogeneous        about 50%);    -   elimination of internal “TATA box” sequences, a portion rich in        AT or GC, elements with sequences ARE, INS and CRS, repeat        sequences, sequences with secondary RNA structure, cryptic        splicing sequences;    -   elimination of the following restriction sites: NheI, BamHI,        XhoI, EcoRI, KpnI, SalI, BspEI, BgIII, NotI, BssHII, BsiWI, with        the exception of BsiWI in the first position and BssHII in the        last position;    -   Elimination of TTTT, TTTAA, AAAGGG, AAAAGG, GGGAAA, GGGGAA        motifs and their complements TTCCCC, TTTCCC, CCTTTT, CCCTTT.

TABLE 1 Antibody response directed against the MVSchw virus in CD46⁺/IFNAR mice inoculated i.p. with MV_(Schw)-[EDIII]_(DV-1) ImmunizationMV Ag DV-1 Ag DV-1 rEDIII Anti-DV-1 (month) titer^(c) titer^(d)titer^(e) FRNT75^(f) 1^(a) 15000 <100 <100 ND 2^(b) 40000 100 10 <10^(a)10⁴ TCIP₅₀ of MV-DV-1 [EDIII + M¹⁻⁴⁰]_(DV1)was given i.p. to fouradult mice; ^(b)The individuals received a booster injection with 10⁴TCIP₅₀ of MV-DV-1 [EDIII + M¹⁻⁴⁰]_(DV-1;) ^(c)Determined by ELISA(Trinity Biotech) on pooled serums inactivated by heating;^(d)Determined by ELISA on pooled serums inactivated by heating.Microtitration plates were coated with 5 × 10⁵ FFU of purified sucrose,FGA/NA d1d as viral antigen; ^(e)Determined by ELISA on pooled serumsinactivated by heating. Microtitration plates were coated with 50 ng ofhighly purified recombinant DV-1 EDIII as viral antigen; ^(f)Anti-DV-1neutralization antibodies were detected using a focus reductionneutralization test (FRNT). Pooled serums inactivated by heating wereincubated with the DV-1 Hawaii line and titrating the virus on Verocells using the plaque immunoassay test. FRNT75, the highest serumdilution tested, reduced the number of FFU by at least 75%.

TABLE 2 Antibody response directed against the MVSchw virus inCD46⁺/IFNAR mice inoculated i.p. with MV_(Schw)-[EDIII +^(M1-)40]_(DV-1) Immunization MV Ag DV-1 Ag DV-1 rEDIII Anti-DV-1(months) titer^(e) titer^(f) titer^(g) FRNT75^(h)  1^(a) 15000 <100 ≦100ND  2^(b) 40000 1600 400 10 3 30000 1000 600 10 6 20000 500 100 40 7^(c) 20000 20000 100000 800 9 10000 2000 10000 100 10^(d ) 10000200000 800000 4000 ^(a)10⁴ TCIP₅₀ of MV-DV-1 [EDIII + M¹⁻⁴⁰]_(DV1)wasgiven i.p. to four adult mice; ^(b)The individuals received a boosterinjection with 10⁴ TCIP₅₀ of MV-DV-1 [EDIII + M¹⁻⁴⁰]_(DV-1;) ^(c)Theimmunized mice received a booster injection of 5 μg of total secretedproteins deriving from a supernatant of S2 cells expressing rDV1[EDIII +M¹⁻⁴⁰]_(DV-1) in the presence of Alugel adjuvant; ^(d)The immunized micewere inoculated i.p. with 10⁷ FFU of FGA/NA d1d from the DV-1 line forthree weeks; ^(e)Determined by ELISA (Trinity Biotech) on pooled serumsinactivated by heating; ^(f)Determined by ELISA on pooled serumsinactivated by heating. Microtitration plates were coated with 5 × 10⁵FFU of purified sucrose, FGA/NA d1d as viral antigen; ^(g)Determined byELISA on pooled serums inactivated by heating. Microtitration plateswere coated with 50 ng of highly purified recombinant DV-1 EDIII asviral antigen; ^(h)Anti-DV-1 neutralization antibodies were detectedusing FRNT. Pooled serums inactivated by heating were incubated with theDV-1 Hawaii line and titrating the virus on Vero cells using the plaqueimmunoassay test. FRNT75, the highest serum dilution tested, reduced thenumber of FFU by at least 75%.

1. A chimeric polypeptide comprising a peptide of a subdomain of the Eprotein of Flaviviridae bound to a peptide of a subdomain of themembrane M protein of Flaviviridae.
 2. The chimeric polypeptideaccording to claim 1, wherein the peptide of the subdomain of the Eprotein consists of the ectodomain III comprising an amino acid sequenceselected from the group consisting of: amino acids 19 to 120 of SEQ IDNO: 1; amino acids 19 to 119 of SEQ ID NO: 3; amino acids 21 to 123 ofSEQ ID NO: 6; amino acids 21 to 121 of SEQ ID NO: 12; and amino acids 21to 123 of SEQ ID NO:
 14. 3. The chimeric polypeptide according to claim1, wherein the peptide of the subdomain of the E protein consists of adimer of the ectodomain III of the dengue 1, 2, 3 or 4 virus comprisingan amino acid sequence as defined in at least one of the followingsequences: amino acids 21 to 262 of SEQ ID NO: 8; and amino acids 21 to262 of SEQ ID NO:
 10. 4. The chimeric polypeptide according to claim 1,wherein the peptide of the subdomain of the E protein consists of atetramer of the ectodomain III of the dengue 1, 2, 3, 4 virus comprisinga sequence ranging from amino acid 21 to 494 of SEQ ID NO: 16 or rangingfrom amino acid 18 to 429 of SEQ ID NO:
 24. 5. The chimeric polypeptideaccording to claim 1, wherein the peptide of the subdomain of the Mprotein consists of the ectodomain 1-40 comprising an amino acidsequence ranging from position 123 to position 170 of SEQ ID NO:
 3. 6.The chimeric polypeptide according to claim 1, wherein the peptide ofthe subdomain of the M protein consists of the apoptoM sequencecomprising an amino acid sequence ranging from position 154 to 170 ofSEQ ID NO: 3 or ranging from position 122 to 132 of SEQ ID NO:
 12. 7.The chimeric polypeptide according to claim 1, further comprising abinding segment binding the peptide of the subdomain of the E protein tothe peptide of the subdomain of the M protein.
 8. The polypeptideaccording to claim 7, wherein the binding segment is a pentapeptidehaving An amino acid sequence RRDKR or RREKR.
 9. The chimericpolypeptide according to claim 1, comprising at least one amino acidsequence selected from the group consisting of: amino acids 19 to 172 ofSEQ ID NO: 3; amino acids 21 to 168 of SEQ ID NO: 6; amino acids 21 to132 of SEQ ID NO: 12; amino acids 18 to 624 of SEQ ID NO: 20; aminoacids 18 to 609 of SEQ ID NO: 21; amino acids 18 to 624 of SEQ ID NO:22; amino acids 18 to 489 of SEQ ID NO: 23; and amino acids 21 to 474 ofSEQ ID NO:
 24. 10. The chimeric polypeptide according to claim 1,wherein the Flaviviridae is selected from the group consisting of WestNile virus, dengue virus, Japanese encephalitis virus and yellow fevervirus.
 11. An isolated polynucleotide coding for a chimeric polypeptideas defined in claim
 1. 12. The polynucleotide according to claim 11,comprising a nucleotide sequence selected from the group consisting of:nucleotides 7 to 492 of SEQ ID NO: 4; SEQ ID NO: 5; nucleotides 7 to 504of SEQ ID NO: 7; nucleotides 7 to 504 of SEQ ID NO: 13; and SEQ ID NOs:25 to
 29. 13. A recombinant measles viral vector comprising apolynucleotide according to claim 11 inserted into the viral genome. 14.The recombinant viral vector according to claim 13, wherein said vectoris a live measles or Schwarz strain viral vector.
 15. The recombinantviral vector according to claim 13, selected from the group consistingof viral vectors constituted by those deposited at the CNCM undernumbers I-3440, I-3442, I-3452, I-3453, I-3454, I-3455, I-3619, I-3620,I-3621, I-3622 and I-3623.
 16. A method for the preparation of animmunogenic composition intended for the prevention or treatment of aFlaviviridae infection in a sensitive species comprising preparing therecombinant vector according to claim 14 and introducing the vector intoa suitable host.
 17. A purified monoclonal or polyclonal antibodyspecifically recognizing at least the chimeric polypeptide defined inclaim
 1. 18. A cloning or expression vector comprising a polynucleotideas defined in claim
 11. 19. An immunogenic composition intended for theprevention and/or the treatment of a Flaviviridae infection in asensitive species comprising at least one selected from the groupconsisting of: a chimeric polypeptide as defined in claim 1; apolynucleotide encoding the chimeric polypeptide of claim 1; arecombinant viral vector comprising the polynucleotide encoding thechimeric polypeptide of claim 1; an antibody specifically recognizing atleast the polynucleotide encoding the chimeric polypeptide of claim 1 orthe polypeptide encoded by the polynucleotide of claim 1; and a cloningand/or expression vector comprising the polynucleotide encoding thepolypeptide of claim
 1. 20. The immunogenic composition according toclaim 19, further comprising a pharmaceutically acceptable vehicle. 21.A method for preventing and/or treating a Flaviviridae infection in asensitive species, comprising administering a pharmaceutically effectiveamount of at least one selected from the group consisting of: a chimericpolypeptide as defined in claim 1; a polynucleotide encoding thechimeric polypeptide of claim 1; a recombinant viral vector comprisingthe polynucleotide encoding the chimeric polypeptide of claim 1; anantibody specifically recognizing at least the polynucleotide encodingthe chimeric polypeptide of claim 1 or the polypeptide encoded by thepolynucleotide of claim 1; and a cloning and/or expression vectorcomprising the polynucleotide encoding the polypeptide of claim
 1. 22.The method according to claim 21, further comprising a boosterimmunization step comprising additional administration of a chimericpolypeptide comprising a peptide of a subdomain of the E protein ofFlaviviridae bound to a peptide of a subdomain of the membrane M proteinof Flaviviridae in the presence of an adjuvant.
 23. The method accordingto claim 21, wherein the Flaviviridae is selected from the groupconsisting of a dengue virus, yellow fever virus, Japanese encephalitisvirus and West Nile fever virus.